Chromosomal aneuploidy is an important cause of morbidity during the prenatal and postnatal life. Assessment of chromosomal aneuploidy has traditionally been associated with the investigation of fetal viability and prenatal diagnosis. Methods for the detection and characterization of chromosomal aneuploidy include karyotyping of metaphase chromosomes, fluorescence in-situ hybridization (FISH) (Homer, J. et al., Prenat Diagn 23:566-571 (2003)), quantitative fluorescence polymerase chain reaction (PCR) (Mann, K. Methods Mol Med 92:141-156 (2004)), gene dosage PCR (Zimmermann, B. et al., Clin Chem 48:362-363 (2002), and array-based comparative genomic hybridization (CGH) (Hu, D. G. et al., Mol Hum Reprod (2004)).
Fetal chromosomal aneuploidy is known to contribute significantly to fetal loss and accounts for 50% of the first trimester spontaneous abortions (Chitty, L. Br Med Bull 54:839-856 (1998)). Viable fetuses are especially associated with certain types of chromosomal aneuploidy. Trisomy 21, or Down syndrome, is the most common autosomal aneuploidy compatible with postnatal survival with an occurrence of 1 in 800 live births (Hook, E. B. Lancet 2:169-172 (1981)). Trisomy 21 is the most common reason why couples opt for prenatal diagnosis. At present, definitive diagnosis of a trisomy 21 fetus and other chromosomal aneuploidies rely on the genetic analysis of fetal genetic material obtained by invasive procedures such as amniocentesis and chorionic villus sampling (CVS). By virtue of their invasive nature, these procedures are associated with a finite risk of spontaneous abortion. Hence, other noninvasive approaches have been developed to stratify pregnancies according to the risk of carrying a trisomy 21 fetus. Only pregnancies with a substantive risk, defined as a risk greater than the procedure-related risk of fetal loss, are recommended to undergo the invasive procedures. The risk stratification strategies that have been in current use include the assessment of maternal age, maternal serum biochemical markers and fetal ultrasound features (Nicolaides, K. H. et al., Prenat Diagn 22:308-315 (2002)).
To attain better sensitivity and specificity, various combinations of markers and approaches have been evaluated (Wald, N. J. et al., Prenat Diagn 17:821-829 (1997)), including the triple test, quadruple test (Wald, N. J. et al., Lancet 361:835-836 (2003)), integrated test (Wald, N. J. et al., N Engl J Med 341:461-467 (1999)) and first trimester screening (Wapner, R. et al., N Engl J Med 349:1405-1413 (2003)). Serum biochemical markers in use include, alpha-fetoprotein, unconjugated estriol, total or free beta-human chorionic gonadotropin, inhibin-A, and pregnancy-associated plasma protein-A (PAPP-A). Pregnancies whose risk is shown to be high by these screening modalities are ultimately referred for amniocentesis or CVS.
Recently, the discovery of circulating cell-free fetal nucleic acids in maternal plasma has provided an alternative source of fetal genetic material which can be sampled noninvasively (Lo, Y. M. D. et al., Lancet 350:485-487 (1997); Poon, L. L. M. et al., Clin Chem 46:1832-1834 (2000)). Furthermore, the concentrations of circulating fetal DNA in the plasma of women carrying trisomy 21 fetuses have been shown to be significantly higher than those in women carrying euploid fetuses (Lo, Y. M. D. et al., Clin Chem 45:1747-1751 (1999); Zhong, X. Y. et al., Prenat Diagn 20:795-798 (2000)). Recently, circulating fetal RNA has also been shown to be promising as a class of gender-independent fetal nucleic acid markers in maternal plasma. (Ng, E. K. O. et al., Clin Chem 49:727-731 (2003); Ng, E. K. O. et al., Proc Natl Acad Sci USA 100:4748-4753 (2003)). Thus, circulating fetal nucleic acid quantification is useful as an additional prenatal screening marker for the risk stratification of pregnancies.
Placenta-expressed mRNA transcripts, such as those coding for human placental lactogen (hPL), human chorionic gonadotropin beta subunit (βhCG) (Ng, E. K. O. et al., Clin Chem 49:727-731 (2003)), corticotropin releasing hormone (CRH) (Ng, E. K O. et al., Proc Natl Acad Sci USA 100:4748-4753 (2003b)), tissue factor pathway inhibitor 2 (TFPI2), KiSS-1 metastasis-suppressor (KISS1) and placenta-specific 1 (PLAC1) (Tsui, N. B. Y. et al., J Med Genet 41:461-7 (2004)), have been shown to be detectable in maternal plasma. These placenta-derived mRNA species are known to be pregnancy-specific (Ng, E. K. O. et al., Proc Natl Acad Sci USA 100:4748-4753 (2003); Tsui, N. B. Y. et al., J Med Genet 41:461-7 (2004)). In particular, aberrant elevations in CRH mRNA concentrations in maternal plasma have been reported in preeclamptic pregnancies (Ng, E. K. O. et al., Clin Chem 49:727-731 (2003)). As these placenta-expressed markers are pregnancy-specific but gender- and polymorphism-independent, they are useful in the noninvasive prenatal assessment of all pregnancies.
Chromosomal aneuploidy alters the dosage of genes located on the aneuploid chromosome. The altered gene dosage can be reflected by a distorted allele ratio of the genes. The distorted allele ratio is in turn reflected by a distorted ratio of alleles of polymorphisms present on the RNA transcript of the genetic loci on the aneuploid chromosome. One example of such polymorphisms is single nucleotide polymorphism (SNP) in which the ratio of the SNP alleles may be distorted in the presence of aneuploidy of the chromosome concerned. Thus, a reference range for the RNA-SNP ratio is established for normal pregnancies and fetal trisomy 21 can be determined when a deviation from the reference ratio is observed. In comparison to conventional cytogenetic methods of analysis, the proposed technique does not require prior culturing of the fetal cells and therefore shortens the analytical time. In addition, maternal blood samples can be obtained non-invasively, thus minimizing potential harm to both the fetus and the mother.